Elimination of Daudi lymphoblasts from human bone marrow using avidin- biotin immunoadsorption

Biotinylated antibodies and an avidin-Sepharose 6MB column were utilized in a novel approach to deplete selected cell populations from human bone marrow. Fluorescein-labeled Daudi lymphoblasts were mixed with bone marrow mononuclear cells in a model system, and removal of Daudi cells was quantitatively assessed with an inverted fluorescent microscope. Treatment using the biotinylated monoclonal antibody 2H7 reactive with Bp32 antigen (expressed on Daudi cells) followed by passage over avidin-Sepharose produced greater than two logs of Daudi cell removal from bone marrow. An alternative method was tested by treating cells successively with nonbiotinylated monoclonal antibody and biotinylated goat antimouse immunoglobulin followed by passage over avidin-Sepharose. Up to three logs of Daudi cells could be eliminated from bone marrow with quantitative recovery of hematopoietic progenitors. The use of biotinylated goat antimouse immunoglobulin eliminates the need to prepare a biotin conjugate of each individual monoclonal antibody. The clinical application of cellular immunoadsorption using the avidin-biotin system may prove useful in bone marrow transplantation.     

Transplantation of allogeneic CD34+ peripheral blood stem cells in patients with advanced hematologic malignancy

Sixteen patients with advanced hematologic malignancies were transplanted with HLA-identical allogeneic peripheral blood stem cells (PBSCs) that were selected for CD34+ cells by an avidin-biotin immunoadsorption technique. The median age of patients was 48 years (range, 37 to 67). Patients received 12.0 or 13.2 Gy of total body irradiation followed by 120 mg/kg of cyclophosphamide. Normal donors received 16 mg/kg of granulocyte-colony stimulating factor on days 1 to 6 followed by PBSC harvests on days 4 to 7. PBSC harvests were processed each day on a single avidin-blotin column containing an antibody to the CD34 antigen and processed cells were infused without cryopreservation daily for 4 consecutive days. Prophylaxis against graft-versus-host disease (GVHD) consisted of cyclosporine alone for 5 patients and CSA plus methotrexate for 11 patients. A median of 18.64 (6.74 to 34.97) x 10(8) CD34+ cells/kg patient body weight were collected from each donor. A median of 8.96 (2.62 to 17.34) x 10(8) CD34+ cells/kg patient body weight were recovered after avidin-biotin adsorption which represented a median CD34+ cell yield of 53% (18% to 77%) with a median purity of 62% (34% to 82%). There was a reduction in CD3+ cells from a median of 557.26 (227.73 to 677.77) x 106/kg to 0.73 x 10(4)/kg (0.40 to 3.65), in CD4+ cells from 351.72 (194.47 to 520.11) x 10(6)/kg to 0.40 (0.15 to 1.03) x 10(4)/kg and in CD8+ cells from 169.74 (53.34 to 325.83) x 10(6)/ kg to 0.32 (0.12 to 2.71) x 10(4)/kg representing a median 2.8 (2.19 to 3.14) log reduction in T cells. One patient died of infection on day 3 posttransplant and was unevaluable for recovery of neutrophils. The median day to recovery of 500 neutrophils/mL was 15 (8 to 26) in the remaining 15 patients. Six of 16 patients falled to achieve a platelet count of 20,000/mL before death on days 3 to 97 of transplant-related complications. The median day to achieving platelets of 20,000 mL in the remaining 10 patients was 11 (7 to 31). Eight of 16 patients (50%) died between 3 and 97 days posttransplant, 7 of transplant-related causes, and 1 of progressive disease. Grade 2-4 acute GVHD occurred in 12 out of 14 (86%) and grades 3-4 in 6 out of 14 (43%) evaluable patients. Six of 8 evaluable patients developed clinical chronic GVHD and 1 developed subclinical chronic GVHD. Bone marrow and/or peripheral blood chimerism studies in 12 evaluable patients showed 97% to 100% donor type in 11 patients with 1 patient in relapse showing 40% donor cells 60 to 90 days posttransplant. Four of 16 patients (25%) are alive and disease-free 312 to 576 days after transplant. There were no episodes of graft failure or rejection. This study shows that allogeneic transplantation using CD34+ selected PBSC results in prompt and sustained engraftment. CD34+ selection, as employed in this preliminary study, however, resulted in an apparently higher rate of acute and chronic GVHD. However, The sample size is quite small and precludes a more definitive conclusion regarding GVHD.     

综合性医院质量管理科职能的探索与实践

本文从医院发展战略和学科建设角度,对综合性医院质量管理科的职能进行探讨,认为综合性医院质量管理科具有建立和完善医院质量管理组织体系、指标体系、控制体系、分析讲评机制、信息交流机制、开展质量教育、引进和推广管理理念和方法等职能。     

激光快速成形技术制作镍铬合金基底冠的实验研究

目的 研究激光快速成形技术制作镍铬合金基底冠的可行性.方法 利用逆向工程和计算机辅助设计技术设计基底冠,应用激光快速成形技术制作镍铬合金基底冠,分别测量基牙预备体代型外表面(牙合)面边缘(A组)、轴面中点(B组)和肩台边缘处(C组)与镍铬合金基底冠内表面的间隙,与临床可接受的标准(120μm)进行单样本t检验.结果 A、B、C组间隙值分别为(82.60±13.58)、(45.80±16.12)、(57.90±9.04)μm,各组均小于120μm,差异均有统计学意义(tA=8.71,tB=14.56,tC=21.72,P＜0.05).结论 利用激光快速成形技术可加工出厚度为0.8 mm、与基牙间隙低于120μm的镍铬合金基底冠.     

胃癌中多基因甲基化状态及其表达的研究

目的了解胃癌中p16、E-cadherin、Runx3、DAPK和CHFR基因启动子CpG岛的甲基化状态,并探讨基因异常甲基化与mRNA表达的关系及其意义。方法采用甲基化特异性PCR(MSP)技术检测12例胃癌患者癌组织及其相应的癌旁正常组织中5种基因的甲基化状态,以12例胃镜活检的正常胃组织作为对照,采用RT-PCR方法相应检测了5种基因的mRNA表达水平。并分析每种基因甲基化程度与表达水平之间的关系。结果胃癌组织中,p16、E-cadherin、Runx3、DAPK和CHFR基因的甲基化率分别为41.7%、8.3%、58.3%、33.3%和58.3%,相应的癌旁正常组织中,p16、E-cadherin、Runx3、DAPK和CHFR基因的甲基化率分别为8.3%、0.0%、8.3%、8.3%和16.7%,且75%的胃癌组织中至少有一种基因甲基化,显著高于癌旁正常组织25%(P<0.05),而健康对照组织中无基因甲基化,在甲基化的胃癌组织中均无p16、E-cadherin和DAPKmRNA表达,而癌旁正常组织、非甲基化的癌组织中均有其表达。结论胃癌中多基因启动子CpG岛高甲基化是一个频繁的事件,并且基因启动子高甲基化与其mRNA表达缺失有关,这可能参与了胃癌的发生发展。     

Tumor-specific aneuploidy not detected in CD19+ B-lymphoid cells from myeloma patients in a multidimensional flow cytometric analysis

Aneuploidy and lg light chain restriction were used as separate, independent tumor specific markers to study 26 patients with multiple myeloma to determine whether bone marrow B cells, as defined by CD19 expression, are clonally related to myeloma plasma cells. Specimens were characterized using multidimensional flow cytometry to identify the presence of clonality in both the B lymphoid and plasma cell populations using both surface and cytoplasmic staining with antibodies specific for kappa or lambda lg light chain In none of the patients with multiple myeloma were CD19+ cells found to be clonally restricted to kappa or lambda. The monoclonal plasma cells (MPC) were found to be uniformly negative for CD10, CD19, and CD34, while the CD19+ B lymphoid cells present within the samples expressed normal intensities and relationships of these antigens, which allowed them to serve as internal positive controls. Combined analysis of call surface antigen expression and DNA content allowed plasma cell populations to be characterized for aneuploidy without interference from normal bone marrow cells. The MPC, detected on the basis of bright CD38 expression (CD38+2), demonstrated DNA aneuploidy in 65% of cases (DNA index range of 0.9 to 1.3). These aneuploid DNA distributions had typical cell cycle profiles (including G1,S and G2+M) expected of a proliferating population. In all cases, DNA aneuploidy was confined almost entirely to the CD38+2, CD19- malignant plasma cells, while cells expressing CD19 were diploid. These results support the concept that myeloma is a disease process mediated by self-replicating, late compartments of B- cell ontogeny.     

Effect of cell concentration on bone marrow and peripheral blood stem cell cryopreservation

The effects of cell concentration during cryopreservation on bone marrow (BM) or peripheral blood (PB)-derived hematopoietic progenitor cells have not been described. The much greater numbers of cells harvested for autologous PB stem cell (PBSC) transplantation requires that the cells be frozen at higher cell concentrations, or in much greater volumes, compared with BM. We cryopreserved 108 PBSC collections from 30 patients at an average (+/- SD) cell concentration of 3.7 +/- 1.9 x 10(8) nucleated cells per mL in 127 +/- 45 mL. The proportion of mononuclear cells was 52.9% +/- 27.2%. The products also contained 2.9 +/- 2.1 x 10(9) platelets/mL and an average red cell proportion of 12.9% +/- 7.2%. The nucleated cell recovery after thawing was 75.4% +/- 13.0%. The nucleated cell concentration during freezing was not predictive for the postthaw recoveries of nucleated cells (P = .38), granulocyte-macrophage colony-forming unit (P = .06) or CD34+ cells (P = .54), or for the viability of mononuclear cells (P = .81). The platelet and red cell concentrations similarly were not predictive for these endpoints. Samples (3 BM, 7 PBSC) from 10 patients were simultaneously cryopreserved at two-fold, and from 5 additional patients (PBSC) at 6- to 24-fold differing cell concentrations. A lower recovery of erythroid burst forming unit was found for samples frozen at higher cell concentrations (P = .04), but no significant differences were found in the other endpoints listed above. The average cell concentration during freezing for each patient's PBSC collections (n = 34 patients) did not predict time to achieve a PB count of > 500 granulocytes/microL (P = .51) or platelet transfusion independence (P = .39). Patients achieved these endpoints of engraftment at medians of 12 and 13 days, respectively. The infusion of these products was generally well tolerated. Similarly, the cell concentration at which BM cells were frozen did not predict for the duration of granulocyte (P = .63) or platelet (P = .36) aplasias for 54 patients undergoing autologous BM transplantation. These data suggest that PBSC or BM cells collected for transplantation may be cryopreserved at very high cell concentrations without loss of engraftment potential or undue infusion-related toxicity.